布鲁氏菌RPA-SYBR Green Ⅰ快速检测方法的建立与应用

Establishment and application of a rapid detection method for Brucella by RPA-SYBR Green Ⅰ

为了建立一种新型、稳定、可普遍应用的布鲁氏菌检测方法,试验将重组酶聚合酶扩增(recombinase polymerase amplification, RPA)方法与SYBR GreenⅠ结合,根据布鲁氏菌bcsp31基因序列保守区设计1对特异性引物和1条标有biotin、dSpacer、C3 Spacer的特异性探针,建立一种可以检测布鲁氏菌的RPA-SYBR-GreenⅠ方法,对该方法的反应条件和反应体系进行优化,并分析敏感性和特异性,并用该方法和ELISA方法对临床样品进行检测。结果表明:试验成功构建出布鲁氏菌RPA-SYBR-GreenⅠ检测方法。该方法的最佳反应条件为手掌中孵育12 min,闭合手掌即可启动RPA扩增;引物与探针的最佳比例为2.5∶1;最低检测限为1×10^(1)cfu/mL,能够特异地检测出布鲁氏菌;对临床样品的检测结果与ELISA方法一致。说明试验建立的布鲁氏菌RPA-SYBR-GreenⅠ检测方法特异性强,敏感性高,检测速度快,操作简单。

In order to establish a new, stable and universally applicable method for the detection of Brucella, the experiment used the method of recombinase polymerase amplification(RPA) combined with SYBR Green Ⅰ;a pair of specific primers and a specific probe labeled biotin, dSpacer, C3 Spacer, were designed according to the conservative region of Brucella bcsp31 gene sequence. A RPA-SYBR Green Ⅰ method for the detection of Brucella was established;optimize the reaction conditions and the reaction system of this method;the sensitivity and specificity of the method were analyzed. The method and ELISA were used to detect clinical samples. The results showed that the test successfully constructed the Brucella RPA-SYBR Green Ⅰ detection method;the best reaction conditions for this method were 12 min incubation in the palm, and the RPA amplification could be initiated by closing the palm. The best ratio of primer to probe was 2.5∶1;and the lowest detection limit of RPA-SYBR Green Ⅰ method was 1×10^(1)cfu/mL, which could specifically detect Brucella. The results of RPA-SYBR Green Ⅰ method on clinical samples were consistent with ELISA method. The results suggested that the established Brucella RPA-SYBR Green Ⅰ detection method had strong specificity, high sensitivity, fast speed and easy operation.